Features of the response to subchronic low-dose exposure to copper oxide nanoparticles in rats

Copper is an essential trace element for human health and, at the same time, a major industrial metal widely used both in its elemental form and in compounds. We conducted a dose-dependent assessment of the response of outbred albino male rats to subchronic low-dose exposure to copper oxide nanoparticles administered intraperitoneally at cumulative doses of 18 and 36 mg/kg during 6 weeks to exposure groups 1 and 2, respectively. We observed disorders at different levels of organization of the body in the exposed animals, from molecular to organismal. The observed decrease in the activity of succinate dehydrogenase in nucleated blood cells gave evidence of impaired bioenergetics processes. In view of the results of the metabolomics analysis, we assume mitochondrial damage and contribution of apoptotic processes to the pathology induced by copper poisoning. We also assume neurodegenerative effects based on the assessed morphological parameters of the nervous system, results of behavioral tests, and a decreased level of expression of genes encoding NMDA receptor subunits in the hippocampus. The hepatotoxic effect noted by a number of metabolomics-based, biochemical, and cytological indicators was manifested by the impaired protein-synthesizing function of the liver and enhanced degenerative processes in its cells. We also observed a nephrotoxic effect of nanosized copper oxide with a predominant lesion of proximal kidney tubules. At the same time, both doses tested demonstrated such positive health effects as a statistically significant decrease in the activity of alkaline phosphatase and the nucleated blood cell DNA fragmentation factor. Judging by the changes observed, the cumulative dose of copper oxide nanoparticles of 18 mg/kg body weight administered intraperitoneally approximates the threshold one for rats. The established markers of health impairments may serve as a starting point in the development of techniques of early diagnosis of copper poisoning.


Results
Integral indicators of toxicity. Changes in integral indicators of the general toxic effect of exposure to copper oxide nanoparticles were dose-dependent ( Table 1). The duration of the first entry into the dark chamber in the light-dark box test decreased while the summation threshold index describing the ability of the nervous system to sum up subthreshold impulses increased. No changes in the body weight of the animals or their organs (in g/100 body weight) were registered.
Under effect of copper oxide nanoparticles, the activity of succinate dehydrogenase (SDH) in blood lymphocytes showed a statistical decrease in both exposure groups whereas plateletcrit demonstrated a dose-related increase and the absolute platelet count tended to grow with the increasing dose of CuO NPs.
We observed changes in biochemical blood serum parameters, including a decrease in the albumin/globulin ratio owing to a decrease in the absolute number of albumins and a slight increase in globulins, a decrease in the activity of alkaline phosphatase (AP), and an increased activity of alanine aminotransferase (ALT). Imprint cytology. Dose-dependent changes in the ratio of certain types of cells in imprint smears of liver and kidneys following administration of various doses of copper oxide nanoparticles are shown in Table 2. We observed increased numbers of degenerated hepatocytes in liver, degenerated cells of proximal and distal tubules and eosinophils in kidneys.
Electron microscopy. Samples were taken from three animals of each exposure group (n = 6). We examined 108 micrographs of the liver made by electron microscopy.
However, the Pearson's chi-squared goodness of fit test (χ2) compared distribution of mean group values regardless of their intragroup variability. Pairwise comparison of the percentage of each of the morphotypes in the exposure and control groups showed no significant differences in the Mann-Whitney U test (Table 3).
Thus, we noted a tendency to manifestation of cytotoxicity at the level of mitochondrial ultrastructure, namely: a slight decrease in the proportion of normal and an increase in damaged mitochondria across the entire spectrum with the exception of vesicular swollen ones.

Metabolomics activity screening.
Results of the principal component analysis showed the absence of sample clustering in the control group. The observed divergence of samples in the exposure groups was dose dependent (Fig. 2). Moreover, the Student's t-test demonstrated that the number of metabolites that significantly changed their content was significantly higher in the second (higher dose) exposure group.
The statistical analysis of data using the principal component method and the multiple comparison-adjusted t-test in the Mass Profiler Professional software revealed statistically significant changes in both exposure groups (Fig. 3).
Only a part of the selected substances had sufficient intensity of the analytical signal to obtain informative fragment spectra, so the annotation was not possible for all metabolites in the groups. Table 4 shows the list of annotated metabolites.
The changes were likely to be dose-dependent. The level of lysophosphatidylcholines (LPC) decreased whereas those of LPE, lysophosphatidylinositols (LPI), and lysophosphatidylserine (LPS) increased. The level of monoglycerides and palmitic acid decreased and those of methyl hexadecanoic acid and methyl linoleate increased.   Gene expression analysis. Table 5 shows data on genetic alterations in the exposed animals. The highest of the doses tested caused a decrease in the expression of GRIN1 gene in the hippocampus of the experimental rats while that of GRIN2A and GRIN2B genes demonstrated a dose-dependent decline.

Discussion
We have already studied subchronic toxicity of CuO nanoparticles following repeated intraperitoneal injections in rats at the doses of 10 mg/kg b w. 12 and 2.5 mg/kg bw. 13 per day. We observed hematological and biochemical changes in blood, histological changes in liver and kidneys, accumulation of copper in kidneys, and neurodegenerative disorders in the study with a high dose of copper. Subsequent experiments with a lower dose showed a decrease in specific manifestations of copper poisoning. The doses chosen for this study are interesting in terms of the search for more subtle changes at the metabolome and genome levels induced by copper oxide nanoparticles.
Suppression of bioenergetics processes. Mitochondria are known targets for almost all types of damaging agents, including toxins 22 and oxidative stress 23 . A change in the potential of the mitochondrial membrane under effect of copper nanoparticles has been reported 6 . Mitochondrial dysfunction is often difficult to measure and prove 24 ; yet, it can be assumed by alterations in certain markers. Succinate dehydrogenase (SDH) is a mitochondrial enzyme attached to its inner membrane. It is involved in the tricarboxylic acid cycle and the electron transport chain in mitochondria by linking these two processes 25 . SDH dysfunction is of great importance in medical practice as it causes a wide specter of disorders: from neurogenerative 26 to carcinogenic 27,28 , by disrupting mitochondrial functioning and reducing the bioenergetic potential of the cell.
The exposure to CuO nanoparticles caused a decrease in SDH in exposure groups 1 and 2 by 11.95 % and 11.64 %, respectively. In our previous experiments, we also registered a decrease in the activity of this enzyme by 20.72 % at a single dose of 2.5 mg/kg 13 and by 12.23 % at a single dose of 10 mg/kg 12 .
Based on the assumption that functional changes in mitochondria related to their disruption can be detected by electron microscopy, we searched for alterations at the subcellular level. An ultrastructural study of the liver showed a decline in the proportion of normal type A mitochondria. At the same time, such an effect was not observed for the sum of types A and B, both of which could be attributed to normally functioning mitochondria.
Early signs of toxicity. The functions of mitochondria are diverse; they include oxidative phosphorylation for the production of cellular adenosine triphosphate (ATP), involvement in ion homeostasis, some metabolic pathways, programmed cell death, production and consumption of reactive oxygen species 29 . Changes in mitochondria representing cellular "energy stations" are important for the development of pathological conditions of organs and systems and of the whole organism later on.
The metabolomics-based blood testing of the exposed animals showed a relatively slight increase in the level of lysophosphatidylserine (LPS (20:4)), a metabolite that triggers inflammatory processes and acts as a signaling substance in the process of the uptake of apoptotic cells by macrophages 30 . This finding may indicate both the inflammatory process and enhanced apoptosis in the exposed animals.
Apparently, if the inflammatory process does exist, then it is extremely insignificant, since the general blood test results showed no statistical differences between the cases and controls in terms of inflammatory processes, except for the platelet count (Table 1). Platelets serve as an important link between the coagulation and immune systems 31 . A statistically significant increase in the plateletcrit and a trend towards an increase in the number of platelets may indicate inflammation related to production of reactiveoxygen species (ROS) induced by the exposure to CuO NPs 32,33 . ROS, in their turn, are mediators of activation of the immune system 34 . A slight increase in the level of globulins in exposure group 2 was likely to occur owing to an increase in fractions of alpha-1-globulin and beta globulin for the same reason.
A 1.8-fold increase in the eosinophil count indirectly indicates the ability of nanosized copper oxide to stimulate immunocompetent cells, as demonstrated by Tulinska et al. 35 .
In view of the above, we assume contribution of apoptotic changes to the developing pathology. Apart from the increase in the level of lysophosphatidylserine, apoptosis was evidenced by a decrease in the proportion of normal mitochondria and an increase in all types of damaged mitochondria, except for vesicular swollen ones (Table 3). Data on the topology of the inner membrane correlated with the physiological state of mitochondria in the course of cell death, i.e. with the loss of the potential of the mitochondrial membrane, which could be accompanied by a release of cytochrome. We established that the vesicular configuration was associated with the release of proteins and cytochrome 36,37 . Further transition of mitochondria to the swollen configuration occurs only after the loss of potential of the mitochondrial membrane, a long time after the release of cytochrome 38 .
Effects on select organs and systems. Mitochondria are one of the key components of apoptosis in mammalian cells 39 . A relationship between abnormal apoptosis and certain diseases, including neurodegen- Effects on the nervous system. Ionotropic glutamate NMDA receptors are responsible for fast synaptic transmission and play an important role in regulating duration of the excitatory potential 41,42 . In combination with a decrease in the brain weight, we may assume neurodegenerative conditions in the exposed animals (Table 5). This supposition was confirmed by the results of behavioral testing: a rising summation threshold index indicated the predominance of inhibitory processes in the nervous system 43 . An increased anxiety level related to the nanoparticle exposure was judged by a decrease in the duration of the first entry into the dark chamber in the light-dark box test 18,19 . Hepatotoxic effects. We have also found signs of liver damage, presumably mediated by excessive apoptosis. The metabolomics screening showed a decrease in the level of lysophosphatidylcholines, previously noted following the exposure to ions of some heavy metals, including copper 44,45 . Since these substances are synthesized in liver, a decrease in their blood levels can be attributed to an impaired synthetic function of this organ in relation to LPC. This impairment was also proven by a lower albumin to globulin ratio due to a lower albumin count attributed either to suppression of its synthesis or its increased losses, exemplified by a renal disorder accompanied by proteinuria 46 . Yet, we found a decrease in the level of urinary total protein suggesting some suppression of albumin synthesis in liver under effect of CuO NP exposure (at a dose of2 mg/kg). We have described similar suppression of the protein synthesizing function following the exposure to CuO NPs at a single dose of 2.5 mg/kg elsewhere 13 .
We noted a significant decrease in the activity of alkaline phosphatase (AP) in the second exposure group at a single dose of 2 mg/kg. We observed a similar effect of exposure to CuO NPs at a single dose of 2.5 mg/ kg 13 , but not at 10 mg/kg 12 ; besides, it is a classic symptom of the Wilson's disease, a congenital defect of copper metabolism leading to its accumulation 47 . A lower AP activity is associated with zinc and magnesium deficiency since both metals are part of this enzyme 48 . Yet, an impaired AP activity was observed at a subchronic exposure to ZnO NPs 13 , enabling us to assume a key role of the zinc to magnesium ratio in the AP activity. Further studies are necessary to clarify the causes and mechanisms of reducing alkaline phosphatase activity.
A change in the secretory function of the liver was also evidenced by a lower (compared to controls) number of triglyceride cleavage products, i.e. monoglycerides MG(16:0) and MG(18:0), palmitic acid, as well as a higher level of esters of hexadecanoic and linoleic acids. Triglycerides and fatty acid esters are cleaved by hepatic lipase synthesized in this organ and released in the bloodstream; as a result, a decrease in the secretion of this enzyme is expected, which is also supported by an increase in the level of lysophosphatidylethanolamine LPE(20:4), the direct effect of which on liver cells inhibited the expression of genes responsible for the synthesis of lipases 49 . At the same time, indicators of the blood lipid profile of the exposed animals (triglycerides, cholesterol, low and high-density lipoproteins) were not significantly different from those in the controls.
The activity of alanine aminotransferase (ALT) has changed, which may also indicate liver damage in the exposed groups. Changes at the molecular level and in biochemical blood parameters were supported by cytological ones: after the exposure to copper oxide nanoparticles, the number of degenerated hepatocytes in imprint smears of liver increased (Table 2) similar to the proportion of prokaryotic liver cells (Fig. 4C). In addition, we observed marked dystrophic and necrobiotic changes in hepatocytes (Fig. 4B) in the exposed animals compared with the controls (Fig. 4A).

Renal effects.
The nephrotoxic effect of CuO NPs was analyzed by the cytology image of kidney imprint smears ( Table 2). It is known that proximal tubules are damaged to a greater extent than the distal ones 50 owing to a different functional load on these parts. According to our findings presented in Table 2 and those described elsewhere 51 , this statement is also relevant for the effects of nanoparticles.
A statistically significant increase in the eosinophil count was observed in the imprint smears of kidneys in both exposure groups. Our findings are consistent with those published by Cho et al. 52 , indicating the ability of copper oxide nanoparticles to induce inflammation involving eosinophils. It is worth noting the potential role of eosinophils in additional damage to kidney cells: activated eosinophils may induce oxidative stress causing cell death 53 .
Histologically, we observed such pronounced morphological changes as dystrophy of renal cells, tubular luminal dilation, and foci of destruction of the PAS-positive brush border of the tubular epithelium (Fig. 4E) compared with the controls (Fig. 4D). The morphometric analysis showed the loss of the brush border of the renal tubular epithelium (Fig. 4F).
Impact on the genomic DNA fragmentation factor. Previously, the effect of subchronic toxicity of nanosized copper oxide at the genetic level was demonstrated, manifested by a slight but statistically significant increase in the genomic DNA fragmentation factor by 7.58% in liver cells, by 24.66% in spleen cells 12 , and by 2.12% in nucleated blood cells of rats 13 compared to the controls. The ability of nanosized copper oxide to damage DNA have been also confirmed by other studies. It has been demonstrated on the HUVEC cell culture, for instance, that copper ions released from nanosized copper oxide can induce oxidative stress and activation of p38 MAPK signaling pathway and cause subsequent DNA damage and cell death 33 . Their anticancer activity is also closely related to ROS generation 54 .
Against the background of an extensive evidence base, it seems paradoxical that, in contrast to the increase in the genomic DNA fragmentation factor almost always observed by us in experiments with nanoparticles of  12,13 , in the present study we observe more of a protective effect of the latter. This effect can be determined by the fact that modern toxicology has accumulated evidence of nonlinear patterns of the dose-response relationship in addition to classical dependencies. According to Erofeeva (2014), under effect of various pollutants, including heavy metals, the majority (85.7%) of dose-response relationships were precisely nonlinear. At the same time, hormesis was noted in 5.5% of cases whereas the rest 94.5% were paradoxical effects, of which multiphase dependencies similar to vibrational ones prevailed 55 .

Conclusions.
Our findings indicate that subchronic exposure to copper oxide nanoparticles at doses of 1 and 2 mg/kg body weight instilled thrice a week during 6 weeks has a toxic effect at the systemic and organismic levels, although not always dose-dependent. We revealed dose-dependent changes in the rats when assessing the intensity of cellular energy processes, behavioral reaction in rats expressed in the predominance of inhibitory process and a higher anxiety level, and plateletcrit.
The hepatotoxic effect noted by a number of metabolomics-based, biochemical, and cytological indicators was manifested by the impaired protein-synthesizing function of the liver and enhanced degenerative processes in its cells. We also observed a nephrotoxic effect of nanosized copper oxide with a predominant lesion of proximal kidney tubules.
A dose of copper oxide nanoparticles equaling 1 mg/kg body weight administered intraperitoneally 18 times during 6 weeks can be considered as that approximating the threshold one.
The established markers of health disorders may serve as a starting point in the development of techniques of early diagnosis of copper poisoning.

Methods
Preparation of suspension and physicochemical characteristics of copper oxide nanoparticles. The suspension of copper oxide nanoparticles was prepared at the Ural Federal University. We used pulsed laser ablation of thin metal sheet targets of 99.99% pure copper in sterile deionized water. The mean diameter of the suspended copper oxide nanoparticles was 21 ± 4 nm (Fig. 5), as shown by scanning electron www.nature.com/scientificreports/ microscopy (SEM). The suspension stability was judged by the zeta potential measured using the Zetasizer Nano ZS size analyzer (Malvern Panalytical, UK) and was found to be high (up to 42 mV, deionized water), enabling us to increase the particle concentration to 0.25 mg/mL by partial water evaporation at 50 °C without changing the size and chemical identity of nanoparticles.
Experimental animals. The study was conducted on outbred albino male rats, 12 animals in each exposure group. The initial body weight of the animals was 200-270 g with the range within ± 20% of the mean weight. The age of the rodents at the beginning of the experiment was 12-14 weeks out of concerns for a low survival rate of young adults in this subchronic toxicity study of presumably very hazardous nanoparticles. The rats were kept in a specially equipped vivarium room in compliance with the International Guiding Principles for Biomedical Research Involving Animals developed by CIOMS and ICLAS (2012). They were euthanized by rapid decapitation with isoflurane anesthesia used additionally for painless beheading. The choice of the exposure dose was determined by the results of a literature search showing that intraperitoneal instillation of copper oxide nanoparticles at the doses of 5 to 25 mg/kg bw. per day for nine days caused liver and kidney changes and even tissue necrosis 18 . Oral administration of 5 mg/kg bw. of CuO NPs per day for 14 days to Wistar rats induced significant alterations in antioxidant enzyme activities and liver toxicity 15 . Besides, we relied on our own previous experience showing that 10 mg/kg bw. of these NPs significantly altered various indices of the organism 12 while 2.5 mg/kg bw. had no effect 13 .
The intraperitoneal route of administration were chosen for greater accuracy in individual dosing.
Exposure assessment. On the fifth week of exposure, we registered the summation threshold index 56 and conducted a light-dark box test 43,56,57 . After exposure cessation, we visually examined macroscopic changes in internal organs and weighed them; we also analyzed biochemical parameters of blood serum. The bioenergetic state of the rats was determined by the activity of succinate dehydrogenase (SDH) established cytochemically using nitro blue tetrazolium chloride and expressed as the number of formazan granules per 50 cells using optical microscopy with immersion oil 58 . Blood parameters were determined using a Mythic 18 hematology analyzer.
Imprint cytology. For imprint cytology, imprint smears were made from cross-sections of liver and kidneys, dried at the room temperature, and then stained by Leishman stain. The cellular composition and signs of cell damage were analyzed using a Carl Zeiss Primo Star light binocular microscope with a USCMOS video camera visualization system at 100 × and 1000 × magnifications. During microscopy, we counted 100 cells from each smear of lymph nodes and 300 cells from smears of other organs.
Histological studies. Histological specimens were prepared by immersing kidneys and liver in formalin, then cutting them into 2-3 mm thick slices treated with alcohols of increasing concentration, and embedding in www.nature.com/scientificreports/ paraffin. Then, 3-4 μm sections were cut from the embedded blocks and stained with hematoxylin and eosin; in addition, the method of periodic acid for kidneys -Schiff stain was also used. The study of histological preparations, their microphotography and morphometry were carried out using the Avtandilov ocular measuring grid and a computer software for pattern recognition 59-61 using an Olympus CX-41 microscope with an Olympus Soft Imaging Solution GMBH, Model LC20 camera and LCmicro software. At least 30 measurements of each indicator were taken on preparations of four rodents from each exposure and control groups.
Electron microscopy. Cell ultrastructure was assessed using scanning transmission electron microscopy (STEM). The degree of damage to mitochondria was determined according to the classification by Mei G. Sun based on morphological characteristics, such as matrix space and the number of cristae 36 . In calculations, mitochondria of types A (normal) and B (normal vesicular) were counted as normal whereas those of types C (vesicular), D (vesicular swollen), and E (swollen) were considered abnormal.
Metabolomic screening. Metabolomic screening was performed using high performance liquid chromatography-mass spectrometry (HPLC-MS) with a time-of-flight mass spectrometer. Raw data was processed with Agilent MassHunter software package for mass spectra processing and feature extraction. Data processing was performed in Mass Profiler Professional; the statistical analysis included PCA and paired t-test. For each exposure group, we obtained a set of m/z values, which intensity altered statistically during the experiment. Those masses were annotated by exact mass and fragment spectra analysis using available databases (HMDB, MoNA, METLIN, MassBank EU) and in silico fragmentation tools (MetFrag, CFM-ID, MS-FINDER).
Degree of genomic DNA fragmentation. The degree of genomic DNA fragmentation was assessed in the Central Research Laboratory of the Ural State Medical University, Yekaterinburg, Russian Federation, by analyzing amplified fragment length polymorphism on nuclear cells of circulating blood as described elsewhere 62 .
Gene expression analysis. To analyze gene expression, a part of the rat hippocampus was fixed in Intac-tRNA to stabilize RNA in bioassays with the following phenol/chloroform RNA extraction. The quantitative RNA analysis was performed using a Qubit™ 4 fluorometer and the Qubit™ RNA BR Assay Kit. RNA samples were treated with RNase-free DNase I followed by a reverse transcription reaction with a MMLV-RT kit using a SimpliAmp™ Thermal Cycler. Determination of expression of GRIN1, GRIN2A, and GRIN2B genes encoding GluN1, GluN2A, and GluN2B proteins, respectively, was carried out by real-time polymerase chain reaction (RT-PCR) with primers, probes 63 , a ready-made mixture qPCRmix-HS by Evrogen, using a Quant StudioTM 3 RT-PCR system. The level of gene expression was established by the level of mRNA of the studied gene relative to that of the reference "housekeeping" GAPDH gene using the delta delta CT (ΔΔCt) method.
Statistical data processing. The statistical significance of data on behavioral reactions, the genomic DNA fragmentation factor, and biochemical parameters was assessed using the Student's t-test (p ≤ 0.05). In addition, we compared p values estimated by the Student's t-test and the Mann-Whitney test, and their general coincidence proved the appropriateness of applying the t-test. The statistical SEM data processing was performed using the Statistica software by StatSoft. The significance of differences between groups was determined using the Student's t-test, Mann-Whitney U-test, and a one-and two-way ANOVA.
For statistical processing of data related to gene expression, a nonparametric Kruskal-Wallis test was used for pairwise comparison of several groups in the Statistica 12 software.
The difference between mean values was considered statistically significant if the probability of its random occurrence was less than or equal to 0.05 (p ≤ 0.05).
Ethical approval. All methods were carried out in accordance with relevant guidelines and regulations. The

Data availability
The data presented in this study are available on request from the corresponding author.